A photoaffinity probe that targets folate-binding proteins.

Folate and related derivatives are essential small molecules required for survival. Of significant interest is the biological role and necessity of folate in the crosstalk between commensal organisms and their respective hosts, including the tremendously complex human distal gut microbiome. Here, we designed a folate-based probe consisting of a photo-crosslinker to detect and quantitate folate-binding proteins from proteomic samples. We demonstrate the selectivity of our probe for the well-established human folate-binding protein dihydrofolate reductase and show no promiscuous labeling occurs with human caspase-3 or bovine serum albumin, which served as negative controls. Affinity-based enrichment of folate-binding proteins from an E. coli lysate in combination with mass spectrometry proteomics verified the ability of our probe to isolate low-abundance folate-dependent proteins. We envision that our probe will serve as a tool to elucidate the roles of commensal microbial folate-binding proteins in health and microbiome-related diseases.

Evaluation of Folate Receptor-Mediated Cervical Dyeing as a Method for Detection of Cervical Lesions.

OBJECTIVES: The aim of the study was to evaluate the performance of a folate receptor-mediated tumor detection (FRD) assay for detection of cervical high-grade lesions. MATERIALS AND METHOD: A total of 1504 patients with abnormal cytology and/or positive human papillomavirus (HPV) testing during primary screening from November 2014 to August 2015 were enrolled. The patients were recruited from the Peking University People's Hospital and 12 other hospitals. Folate receptor-mediated tumor detection was applied in all the patients before colposcopy to compare the detection rate, sensitivity, specificity, positive predictive value, negative predictive value, and coincidence rate with HPV and cytology tests according to the pathologic diagnosis. RESULTS: In the total of 1504 patients, 503 patients were negative for intraepithelial lesion or malignancy, 440 patients were cervical intraepithelial neoplasia (CIN) 1, 254 patients were CIN 2, 257 patients were CIN 3, 46 patients were squamous cell carcinoma, and 4 patients were adenocarcinoma in situ. The sensitivity of FRD was 77.72%, which was less than cytology (80.39%) and HPV testing (95.54%). The specificity of FRD was 60.02%, which was greater than cytology (30.12%) and HPV testing (14.95%). The coincidence rate of FRD to the pathologic diagnosis (66.62%) was also significantly greater than atypical squamous cells of undetermined significance cytology and above (48.87%) and HPV testing (45.01%, p < .0001). The detection rate of FRD for all grades of lesions increased with the severity of lesions. CONCLUSIONS: Folate receptor-mediated tumor detection has a slightly lower sensitivity and a higher specificity than cytology and HPV testing for detection of CIN 2+. Simplicity of FRD requires less professional skill. Folate receptor-mediated tumor detection could be a candidate test for cervical cancer screening especially in low- and middle-income countries. However, FRD still needs more clinical trial data to demonstrate its ability in general screening population.

Terminal protection-mediated autocatalytic cascade amplification coupled with graphene oxide fluorescence switch for sensitive and rapid detection of folate receptor.

Folate receptor (FR) is over-expressed in most human tumors and has been regarded as biomarker and therapeutic target. Specific and sensitive detection of FR is essential for tumor treatment and drug development. Here, a specific, sensitive and rapid FR detection strategy was proposed based on terminal protection-mediated autocatalytic cascade amplification coupled with graphene oxide fluorescence switch. Firstly, the specific binding of FR to the folate terminally-labeled on a primary trigger DNA (PT-DNA) could protect the PT-DNA from exonuclease I degradation, converting FR detection to PT-DNA detection. Subsequently, the PT-DNA hybridized with the overhang of 3'-FAM labeled hairpin probe to initiate exonuclease III-assisted hydrolysis, accompanied with the PT-DNA recycling and autonomous generation of secondary trigger DNA and fluorophore. The secondary trigger DNA could as a PT-DNA analogue for the successive hybridization and hydrolysis process, liberating numerous fluorophores within 40min. Finally, The fluorophores kept away from the surface of graphene oxide, achieving significantly amplified fluorescence signal. The specific interaction between FR and folate guaranteed the FR could be distinguished from other proteins with high selectivity. The high fluorescence quenching efficiency of graphene oxide guaranteed a low background. Due to the highly autocatalytic cascade amplification efficiency and low background, sensitive detection of FR had been achieved with the detection limit of 0.44pM. The recoveries from 92% to 107% were achieved by detecting FR in spiked human serum. These results indicate this strategy holds a great potential for reliable quantification of FR in clinical diagnosis and disease treatment.

Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid-desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression.

The reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR) are folate-specific transporters. Antifolates currently in the clinic, such as pemetrexed, methotrexate, and pralatrexate, are transported into tumor cells primarily via RFC. Folic acid conjugated to cytotoxics, a new class of antineoplastics, are transported into cells via FR-mediated endocytosis. To better define the role of PCFT in antifolate resistance, a methotrexate-resistant cell line, M160-8, was selected from a HeLa subline in which the RFC gene was deleted and PCFT was highly overexpressed. These cells were cross-resistant to pemetrexed. PCFT function and the PCFT mRNA level in M160-8 cells were barely detectable, and FR-α function and mRNA level were increased as compared with the parent cells. While pemetrexed rapidly associated with FR and was internalized within endosomes in M160-8 cells, consistent with FR-mediated transport, subsequent pemetrexed and (6S)-5-formyltetrahydrofolate export into the cytosol was markedly impaired. In contrast, M160-8 cells were collaterally sensitive to EC0905, a folic acid-desacetylvinblastine monohydrazide conjugate also transported by FR-mediated endocytosis. However, in this case a sulfhydryl bond is cleaved to release the lipophilic cytotoxic moiety into the endosome, which passively diffuses out of the endosome into the cytosol. Hence, resistance to pemetrexed in M160-8 cells was due to entrapment of the drug within the endosome due to the absence of PCFT under conditions in which the FR cycling function was intact.

Distinguishing folate-receptor-positive cells from folate-receptor-negative cells using a fluorescence off-on nanoprobe.

Based on the high affinity of folic acid (FA) for folate receptor (FR) that is overexpressed on the surface of many human cancer cells, we have developed a simple fluorescence nanoprobe (1) with multiple capability (fluorescence off-on response and cell-targeting ability) for imaging of FR-positive cells by covalently linking both FA and Rhodamine B (RB) to graphene oxide (GO) through disulfide bonds. The nanoprobe shows a weak fluorescence due to the electron transfer from GO to RB. However, the specific binding of FA to FR-positive cells leads to the internalization of the nanoprobe into the cells. As a result, the disulfide bonds of 1 are cleaved by intracellular glutathione, causing the release of the RB moiety from GO and thereby the generation of fluorescence. Compared to most of the reported fluorescence always-on nanoprobes for imaging FR-positive cells, the present fluorescence off-on nanoprobe can not only produce a high signal/background ratio but also avoid the false positive results often caused by nonspecific adsorption of the always-on nanoprobes on the surface of nontarget cells. Notably, the proposed off-on nanoprobe has been demonstrated to distinguish the cells with different expression levels of FR by culturing and analyzing different cell mixtures (Hela/NIH-3T3 and Hela/MCF-7 cells). Moreover, the nanoprobe is capable of discriminating FR-positive from FR-negative cells even with similar morphology. This method is simple and selective for fluorescence imaging of FR-positive cells.